The biological characteristics of bladder cancer include enhanced invasion and migration, which are the main causes of death in patients. Starvation is a typical feature of the bladder cancer microenvironment and can induce autophagy. Autophagy has an important relationship with the invasion and migration of tumors. However, the role of autophagy in the invasion and migration of bladder cancer cells remains unclear. Hence, the aim of the current study was to clarify this role and underlying mechanism. In this study, we found that starvation enhanced the epithelial‐mesenchymal transition (EMT)‐mediated invasion and migration of T24 and 5637 cells while inducing autophagy. The inhibition of autophagy with chloroquine (CQ) or 3‐methyladenine (3MA) decreased EMT‐mediated invasion and migration. In addition, the expression of transforming growth factor 1 (TGF‐β1) and phosphorylated Smad3 (p‐Smad3) increased after starvation. The inhibition of autophagy with CQ or 3MA also decreased the expression of TGF‐β1 and p‐Smad3. The inhibitor of TGF‐β receptor sb431542 also inhibited the invasion, migration, and EMT of T24 and 5637 cells during starvation. Furthermore, recombinant TGF‐β1 induced autophagy and inhibition of the TGF‐β/Smad signaling pathway with sb431542 suppressed autophagy. In summary, our results suggested that autophagy promotes the invasion and migration of bladder cancer cells by inducing EMT through the TGF‐β1/Smad3 signaling pathway. Moreover, autophagy and TGF‐β1 can form a positive feedback loop to synergistically promote invasion and migration. Thus, our findings may provide a theoretical basis for the prevention of invasion and migration in bladder cancer.
Overcoming the chemoresistance of bladder cancer is a pivotal obstacle in clinical treatments. Hypoxia widely exists in solid tumors and has been demonstrated to contribute to chemoresistance through hypoxia-inducible factor 1α (HIF‑1α)-mediated autophagy in several types of cancer. However, it is unclear whether HIF‑1α-mediated autophagy and chemoresistance occur in bladder cancer. The present study demonstrated that HIF‑1α was overexpressed in 20 bladder cancer tissues compared with matched paracarcinoma tissues. Gemcitabine-induced apoptosis during hypoxia was significantly reduced compared with that observed under normoxic conditions. In addition, hypoxia activated autophagy and enhanced gemcitabine-induced autophagy. Combined treatment using gemcitabine and an autophagy inhibitor (3-methyladenine) under hypoxia significantly increased gemcitabine cytotoxicity. Furthermore, it was demonstrated that hypoxia-activated autophagy depended on the HIF‑1α/BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3)/Beclin1 signaling pathway. Suppressing HIF‑1α inhibited autophagy, BNIP3 and Beclin1, as well as enhanced gemcitabine-induced apoptosis in bladder cancer cells under hypoxic conditions. Consequently, the results of the present study demonstrated that hypoxia-induced cytoprotective autophagy counteracted gemcitabine-induced apoptosis through increasing HIF‑1α expression. Therefore, targeting HIF‑1α-associated pathways or autophagy in bladder cancer may be a successful strategy to enhance the sensitivity of bladder cancer chemotherapy.
Bladder cancer is a common malignant tumour worldwide. Epithelial–mesenchymal transition (EMT)-related biomarkers can be used for early diagnosis and prognosis of cancer patients. To explore, accurate prediction models are essential to the diagnosis and treatment for bladder cancer. In the present study, an EMT-related long noncoding RNA (lncRNA) model was developed to predict the prognosis of patients with bladder cancer. Firstly, the EMT-related lncRNAs were identified by Pearson correlation analysis, and a prognostic EMT-related lncRNA signature was constructed through univariate and multivariate Cox regression analyses. Then, the diagnostic efficacy and the clinically predictive capacity of the signature were assessed. Finally, Gene set enrichment analysis (GSEA) and functional enrichment analysis were carried out with bioinformatics. An EMT-related lncRNA signature consisting of TTC28-AS1, LINC02446, AL662844.4, AC105942.1, AL049840.3, SNHG26, USP30-AS1, PSMB8-AS1, AL031775.1, AC073534.1, U62317.2, C5orf56, AJ271736.1, and AL139385.1 was constructed. The diagnostic efficacy of the signature was evaluated by the time-dependent receiver-operating characteristic (ROC) curves, in which all the values of the area under the ROC (AUC) were more than 0.73. A nomogram established by integrating clinical variables and the risk score confirmed that the signature had a good clinically predict capacity. GSEA analysis revealed that some cancer-related and EMT-related pathways were enriched in high-risk groups, while immune-related pathways were enriched in low-risk groups. Functional enrichment analysis showed that EMT was associated with abundant GO terms or signaling pathways. In short, our research showed that the 14 EMT-related lncRNA signature may predict the prognosis and progression of patients with bladder cancer.
Purpose: NUSAP1 has been reported to be involved in the progression of several types of cancer. However, its expression and exact role in bladder cancer (BLCA) remains elusive. The aim of this study was to determine the expression and role of NUSAP1 in BLCA. Methods: Tissue microarray, real-time PCR, Western blot and immunohistochemistry assays were carried out to determine NUSAP1 expression in BLCA tissues and cells. The biological roles of NUSAP1 were investigated using CCK-8, EdU labeling, flow cytometry, Transwell, and wound healing assays. Additionally, the effect of NUSAP1 on epithelialmesenchymal transition (EMT) was investigated by Western blotting and real-time PCR. Results: We found that NUSAP1 was upregulated in BLCA, and its expression was closely related to the poor prognosis of patients. Subsequently, we transfected 5637 and T24 cell lines with NUSAP1 siRNA and an NUSAP1 overexpression plasmid, respectively. NUSAP1 downregulation in 5637 cells inhibited cell proliferation, migration, and invasiveness and enhanced chemosensitivity to gemcitabine, while NUSAP1 overexpression in T24 cells resulted in the inverse effects. Moreover, NUSAP1 regulated EMT via the TGF-β signaling pathway, and when TGF-beta receptor 1 (TGFBR1) was inhibited with the inhibitor SB525334, the invasion and metastasis ability of BLCA cells was significantly suppressed, as well as p-Smad2/3 and vimentin expression. Conclusion: Our above data demonstrate that NUSAP1 contributes to BLCA progression via the TGF-β signaling pathway.
Established studies proved that mechanical compression loading had multiple effects on the biological behavior of the intervertebral disc (IVD). However, the regulating mechanism involved in this process remains unclear. The current study is aimed at exploring the potential bioregulators and signaling pathways involved in the compression-associated biological changes of nucleus pulposus (NP) cells. Tandem mass tag- (TMT-) based quantitative proteomics was exerted to analyze the differentially expressed proteins (DEPs) and signal pathways among the different groups of NP cells cultured under noncompression, low-compression (LC), and high-compression (HC) loading. Eight potential protective bioregulators for the NP cell survival under different compression loading were predicted by the proteomics, among which macrophage migration inhibitory factor (MIF) and oxidative stress-related pathways were selected for further evaluation, due to its similar function in regulating the fate of the cartilage endplate- (CEP-) derived cells. We found that deficiency of MIF accentuates the accumulation of ROS, mitochondrial dysfunction, and senescence of NP cells under overloaded mechanical compression. The potential molecular mechanism involved in this process is related to the mitophagy regulating role of MIF. Our findings provide a better understanding of the regulatory role of mechanical compression on the cellular fate commitment and matrix metabolism of NP, and the potential strategies for treating disc degenerative diseases via using MIF-regulating agents.
Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cell carcinoma and the incidence of this disease is increasing. The present study aimed to investigate the role of homeobox A6 (HOXA6) in the proliferation and apoptosis of ccRCC cells. Analysis of the GSE6344 dataset and immunohistochemistry revealed that the mRNA and protein expression levels of HOXA6 were suppressed in ccRCC tissues. To evaluate the roles of HOXA6 in cell proliferation and apoptosis, ccRCC cell lines (786-O and 769-P) were transfected with plasmids expressing HOXA6, empty vector, short hairpin (sh)HOXA6 and non-targeting shRNA (NC). Cell Counting Kit-8, colony formation and 5-ethynyl-2′-deoxyuridine staining assays were performed to analyze cell proliferation. In addition, Caspase-Glo and terminal deoxynucleotidyl transferase dUTP nick end labeling assays were performed to detect apoptosis. Furthermore, the cell cycle and apoptotic rates of 786-O and 769-P cells were analyzed by flow cytometry. The results demonstrated that, compared with the empty vector group, the proliferation of 786-O and 769-P cells decreased following HOXA6 overexpression; however, compared with the NC group, cell proliferation increased in the shHOXA6 group. The rate of apoptosis of HOXA6-overexpressing cells was increased compared with the empty vector group, while the rate of apoptosis in the shHOXA6 group was reduced compared with the NC group. In addition, flow cytometry demonstrated that upregulated HOXA6 expression levels could inhibit the cell cycle at the G 0 /G 1 phase. Western blotting revealed that the expression levels of phosphoinositide 3-kinase (PI3K), phosphorylated (p)-protein kinase B (Akt), mitogen-activated protein kinase kinase, p-extracellular signal-regulated kinase (ERK) and B-cell lymphoma 2 (Bcl-2) were suppressed in cells overexpressing HOXA6; however, the protein expression levels of phosphatase and tensin homolog, Bcl-2-associated X protein, cleaved caspase-3 and cleaved-poly (ADP-ribose) polymerase were increased compared with the empty vector group. Opposing results were reported for the shHOXA6 group compared with the NC group. In summary, the results demonstrated that HOXA6 suppresses cell proliferation and promotes apoptosis, which may occur via inhibition of the PI3K/Akt/ERK cascade. These findings indicate the role of HOXA6 in ccRCC; however, the underlying mechanism requires further investigation.
Background Aberrant autophagy and preternatural elevated glycolysis are prevalent in bladder cancer (BLCA) and are both related to malignant progression. However, the regulatory relationship between autophagy and glycolytic metabolism remains largely unknown. We imitated starvation conditions in the tumour microenvironment and found significantly increased levels of autophagy and aerobic glycolysis, which both regulated the progression of BLCA cells. We further explored the regulatory relationships and mechanisms between them. Methods We used immunoblotting, immunofluorescence and transmission electron microscopy to detect autophagy levels in BLCA cells under different treatments. Lactate and glucose concentration detection demonstrated changes in glycolysis. The expression of lactate dehydrogenase A (LDHA) was detected at the transcriptional and translational levels and was also silenced by small interfering RNA, and the effects on malignant progression were further tested. The underlying mechanisms of signalling pathways were evaluated by western blot, immunofluorescence and immunoprecipitation assays. Results Starvation induced autophagy, regulated glycolysis by upregulating the expression of LDHA and caused progressive changes in BLCA cells. Mechanistically, after starvation, the ubiquitination modification of Axin1 increased, and Axin1 combined with P62 was further degraded by the autophagy–lysosome pathway. Liberated β-catenin nuclear translocation increased, binding with LEF1/TCF4 and promoting LDHA transcriptional expression. Additionally, high expression of LDHA was observed in cancer tissues and was positively related to progression. Conclusion Our study demonstrated that starvation-induced autophagy modulates glucose metabolic reprogramming by enhancing Axin1 degradation and β-catenin nuclear translocation in BLCA, which promotes the transcriptional expression of LDHA and further malignant progression.
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